Cells perform diverse processes, such as cell division, growth, motility, formation of adhesions, and tissue morphogenesis, under a wide range of mechanical environments. Central to these processes are mechanical forces, which may come from outside the cell or may be generated internally and which are integrated with signaling pathways to guide the cellular process. The cell's macromolecular cytoskeletal machinery, including the actin-based myosin II motors and actin crosslinking proteins, assemble, function and then disassemble in response to these forces and signaling pathways. This dynamic force-responsive assembly provides self-tuning of the machinery, leading to natural positive and negative feedback and further allows mechanical inputs to be converted (transduced) into signaling outputs. Our collective research spans from single molecule to whole cell level functions with an emphasis on how contractile systems operate to drive cytokinesis and motility and to provide mechanosensory functions for the cell. In this application, we propose studies of two major model protein systems that capture key aspects of force-sensitive macromolecular assembly. Substantial published and unpublished data, including quantitative cell imaging combined with mechanical and genetic perturbations and coupled with computational modeling motivate the questions in this proposal. In particular, we aim to determine the molecular basis for force-dependent assembly of the myosin II bipolar thick filament (BTF). In Aim 1, we will determine the compliance within the BTF and then determine how this compliance restricts the activity of the myosin heavy chain kinase, which tracks the assembled BTF and phosphorylates it to promote BTF disassembly. Quantitative imaging will test how these mechanisms allow for force-dependent BTF assembly in vivo. In Aim 2, we will examine different isoforms of the actin crosslinker alpha-actinin which, based on their in vitro measured kinetic properties, are predicted to display different degrees of mechanosensitive sub-cellular accumulation. We will compare the mechanosensitive accumulation of each alpha-actinin isoform (human ACTN1 and ACTN4 as well as amoeboid ACTN). Because computational modeling supports a catch-slip behavior and/or structural cooperativity as the physical basis of mechanosensitive accumulation, we will determine the force-dependent binding lifetimes for each isoform using single molecule methods. In sum, this research effort will decipher key principles of force-dependent cytoskeletal assembly, which guide cellular processes such as cell division, cell motility, stem cell divisions, and tissue morphogenesis and homeostasis.